Acute Monocytic Leukemia Research Articles

697 Binding of gamma-glutamyl transferase to toll-like receptor-4 allows tissue factor activation in monocytes

Abstract Aims Gamma-glutamyl transferase (GGT) is well known to play a key role in the antioxidant processes, however, it also exerts pro-oxidant effects by activating NFkB, a redox-sensitive transcription factor key in the induction of tissue factor (TF) gene expression, the principal initiator of the clotting cascade. GGT may potentially modulate TF expression, an assumption verified by previous studies carried out in human peripheral blood mononuclear cell (PBMC). Quite importantly, TF expression in response to GGT stimulation was independent of its enzymatic properties since those experiments were conducted by using human recombinant (hr)GGT, a wheat germ-derived protein enzymatically inert because of missing post-translational glycosylation site. Thus, GGT may act through a cytokine-like mechanism although the precise determinants of its action and the receptor involved were not defined by those experiments. To assess whether GGT-induced TF stimulation is a consequence of binding to toll-like receptor (TLR)-4 and activation of NFkB, as suggested by results recently obtained in different experimental contexts. Methods PBMCs obtained from healthy donors through a discontinuous Ficoll/Hystopaque density gradient and THP-1 cells, a human monocytic cell line derived from an acute monocytic leukaemia patient, were incubated with hrGGT (0.5 ng/μL for PBMCs and 1 ng/µL for THP-1). LPS-Rs (0.5 ng/µL for PBMCs and 1 ng/µL for THP-1), CLI-095 (3 × 10−6M) and BAY-11-7082 (10−5 M) were used to block TLR-4 receptors, TLR4 signalling and NFkB respectively. TF protein concentration was determined by western blot analysis. TF pro-coagulant activity (PCA) was assessed through the use of Start Max coagulometer and results were expressed in pg/mL after calibration with a standard curve. HEK-Blue hTLR4-positive and HEK-Blue hTLR4-negative cells are used to evaluate the engagement of TLR4 by hrGGT. Results hrGGT increased TF expression in both PBMCs (PCA from 110 ± 70 to 510 ± 43, n = 7, P < 0.01) and THP-1 cells (PCA from 170 ± 64 to 460 ± 80, n = 15, P < 0.001), result confirmed by western blot. In PBMCs GGT-induced TF stimulation was antagonized by LPS-Rs (PCA: −72 ± 17% n = 4, P < 0.01) a TLR-4 antagonist, CLI-095 (PCA: −74 ± 34%, n = 7, P < 0.001) a TLR-4 intracellular antagonist and BAY-11-7082 (PCA: −71 ± 32%, n = 7, P < 0.001), a NF-kB inhibitor. Similar results were obtained in THP-1 cells (LPS-Rs: −76 ± 15%, n = 6, P < 0.01; CLI-095: −100 ± 6,6 %, n = 6, P < 0.01; BAY-11-7082: −100 ± 2,1%, n = 6, P < 0.01). hrGGT activates NFkB in hTLR4-positive HEK cell lines while does not induces effect in TLR4-negative HEK cell lines. Conclusions These data confirm the cytokine-like activity of GGT and its procoagulant effect in PBMCs and THP-1 cells. Furthermore, it is highlighted for the first time the possible role of TLR-4 as the receptor of GGT and NFkB as the involved signal transduction pathway. The GGT-TLR-4 link may provide an explanation to the consistent association between circulating GGT levels and increased risk of acute thrombotic events as well as to the involvement of GGT in the morbid evolution of the silent atherosclerotic plaque in which GGT colocalizes with monocytes and foam cells, the prime sources of TF within the plaque.

Biological Investigation of Amaryllidaceae Alkaloid Extracts from the Bulbs of Pancratium trianthum Collected in the Senegalese Flora.

Amaryllidaceae plants are rich in alkaloids with biological properties. Pancratium trianthum is an Amaryllidaceae species widely used in African folk medicine to treat several diseases such as central nervous system disorders, tumors, and microbial infections, and it is used to heal wounds. The current investigation explored the biological properties of alkaloid extracts from bulbs of P. trianthum collected in the Senegalese flora. Alkaloid extracts were analyzed and identified by chromatography and mass spectrometry. Alkaloid extracts from P. trianthum displayed pleiotropic biological properties. Cytotoxic activity of the extracts was determined on hepatocarcinoma Huh7 cells and on acute monocytic leukemia THP-1 cells, while agar diffusion and microdilution assays were used to evaluate antibacterial activity. Antiviral activity was measured by infection of extract-treated cells with dengue virus (DENVGFP) and human immunodeficiency virus-1 (HIV-1GFP) reporter vectors. Cytotoxicity and viral inhibition were the most striking of P. trianthum’s extract activities. Importantly, non-cytotoxic concentrations were highly effective in completely preventing DENVGFP replication and in reducing pseudotyped HIV-1GFP infection levels. Our results show that P. trianthum is a rich source of molecules for the potential discovery of new treatments against various diseases. Herein, we provide scientific evidence to rationalize the traditional uses of P. trianthum for wound treatment as an anti-dermatosis and antiseptic agent.

AACE2021-A-1057: The Effect of CLA and Il-13 Treatment on Differentiation Properties of Human Leukaemic Cells (THP-1)

The aim of this study was to investigate the effect of IL13 and CLA on differentiation properties of Macrophages to M2 type using an acute monocytic leukemia (THP1) and further measurement of CD206 (MR) and CD163 as a markers of differentiation using FACS analyzer. Also, to investigate whether M2 differentiation is up regulated through the activation of PPARγ, by the addition of its antagonist GW9662 to down regulate CD206 (MR) expression. The human monocytic cell line THP-1 cell was first derived from the peripheral blood of a 1 year old male with acute monocytic leukemia. Cell Culture Manipulation and sub-culture for the expression CD206 (MR) in CLA and IL-13-treated THP-1 cells. THEN CD206 (MR) expression in CLA and IL-13-treated THP-1 cells in the presence of GW9662. In this study the In-vitro differentiation of monocytes to M2 were enhanced by using Conjugated linoleic acid (CLA) isomers and IL-13, The result shows significant increase in MR (M2 marker) expression on the surface of differentiated cells as well there was significant decrease (P>.005) in the expression of CD 163 suggesting no up regulation occur. The expression of M2 markers (CD206) and PPARγ, a nuclear receptor controlling macrophage inflammation, correlate positively. This was clear when cells were treated with the PPARγ antagonist GW9662 (1μM), the amount of CD206 expression was reduced in both (IL13 and CLA). In conclusion the conducted study shows that CLA and IL-13 enhance the ability of Macrophages to differentiate from M1 to M2 thus promote the anti-inflammatory M2 macrophages phenotype also it demonstrated that PPARγ activation induces polarization of monocytes towards a beneficial M2 phenotype suggesting that CLA and IL-13 partly modulate their action through PPARγ activation.

Dauricine Attenuates Vascular Endothelial Inflammation Through Inhibiting NF-κB Pathway.

Endothelial cells are the fundamental components of blood vessels that regulate several physiological processes including immune responses, angiogenesis, and vascular tone. Endothelial dysfunction contributes to the development of various diseases such as acute lung injury, and endothelial inflammation is a vital part of endothelial dysfunction. Dauricine is an extract isolated from Menispermum dauricum DC, a traditional Chinese medical plant that can be used for pharyngitis. In this work, we found that IL-1β-induced overexpression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin was inhibited by dauricine in primary human umbilical vein endothelial cells (HUVECs). Correspondingly, adhesion of human acute monocytic leukemia cell line (THP-1) to HUVECs was decreased by dauricine. Further studies showed that dauricine inhibited the activation of nuclear factor-κB (NF-κB) pathway in HUVECs stimulated with IL-1β. In vivo, dauricine protected mice from lipopolysaccharide (LPS)-induced acute lung injury. In lung tissues, the activation of NF-κB pathway and the expression of its downstream genes (ICAM-1, VCAM-1, and E-selectin) were decreased by dauricine, consistent with what was found in vitro. In summary, we concluded that dauricine could alleviate endothelial inflammation by suppressing NF-κB pathway, which might serve as an effective candidate for diseases related with endothelial inflammation.

Immunomodulatory properties of dilute drugs in human monocyte/macrophages cell line THP-1 as a model of dose-response effects

Immunomodulatory properties of dilute drugs in human monocyte/macrophages cell line THP-1 as a model of dose-response effects

Study on Anti-Acute Mononuclear Leukemia Activity and Mechanism of Lentinus Edodes Derived β-Glucan/ Selenium Nanoparticles Composites

Objective: Acute monocytic leukemia (AMoL) is the M5 subtype of acute myeloid leukemia (AML) which has the characteristics of high degree of malignancy, prone to extramedullary infiltration, poor efficacy of conventional chemotherapy, easy recurrence and so on. Besides, chemotherapy has large side effects and high cost. Therefore, it is urgent to find natural, non-toxic and cheap drugs for the treatment of acute monocytic leukemia. We evaluated the anti-acute mononuclear leukemia activities of selenium nanoparticles embedded in lentinan in vivo and in vitro, and explored the mechanism of how AMoL cells react to it. It is expected to provide a new method and basis for the treatment of acute mononuclear leukemia.Methods: Utilizing the aggregation and assembly behavior of Lentinus edodes extracted rigid tri-helical β-glucan (Lentinan, LNT) in aqueous solution, cooperative research group prepared Lentinus β-glucan/Selenium nanoparticle complex called LNT-Se by encapsulating selenium nanoparticles (SeNPs) in the cavity of LNT. CCK-8 and flow cytometry were used to detect cytotoxicity, apoptosis rate and cell cycle changes after LNT-Se treatment. Single cell microfluidics chip was used to observe the fluorescence intensity of green fluorescent protein (GFP) of single cell treated by drug. Transmission electron microscopy (TEM) was used to detect ultrastructural changes of THP-1. Coumarin 6 (C6) was used to modify LNT-Se, flow cytometry and confocal microscopy were used to detect drug uptake and primary targeting organelles in AML-M5 cells treated with C6-LNT-Se. The changes of reactive oxygen species (ROS) levels in AML-M5 cells after LNT-Se treatment were detected by flow cytometry. Oxidation-related indexes were further detected, such as malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px). The RNA extracted from untreated and LNT-Se-treated THP-1 cells was sequenced by mRNA-seq. AML-M5 patient-derived tumor xenograft (PDX) leukemia mouse model was established to evaluate the efficacy of LNT-Se in vivo. Serum was collected for detecting liver and kidney functions. Tissue sections were stained by hematoxylin-eosin staining (HE). Specific molecules on the surface of human leukemia cells were detected by immunohistochemistry (IHC) to investigate the efficacy of LNT-Se.Results: SeNPs were successfully embedded into LNT hollow nanotubes and were stably dispersed in water. LNT-Se could significantly inhibit the proliferation of AML-M5 cell lines and had no obvious toxic effect on normal cell lines. LNT-Se could promote the apoptosis of AML-M5 cells and block the cell cycle in the G2/M phase. 50μg/ mL LNT-Se could promote more than 40% apoptosis rate in AML-M5 patient cells, while only about 10% in healthy volunteer cells. At the single cell microfluidic level, the green fluorescence of THP-1 GFP + cells decreased rapidly after 2mg/mL LNT-Se treatment for 2h. After LNT-Se treatment, THP-1 cells showed obvious apoptotic vacuolation, nuclear condensation, chromatin agglutination under TEM. AML-M5 cells could take up C6-LNT-Se and reached the highest uptake at 2h, and C6 fluorescence sites mainly overlapped with lysosomal fluorescence. LNT-Se could decrease intracellular ROS level and MDA content, and increase the activity of GPX in supernatant, but the MDA content and SOD activity in supernatant had no significant changes. Differential gene analysis of transcriptome showed that the first 12 genes were all mitochondrial coding genes. Enrichment analysis showed that TNF, MAPK and NF-κB pathways were up-regulated and oxidative phosphorylation pathways were down-regulated. The experimental results of leukemia PDX animal model showed that the weight loss of mice was slowed down after LNT-Se treatment, and the tumor burden was reduced. Compared with normal mice, there was no significant difference in liver and kidney function after LNT-Se administration, and no pathological changes were observed in viscera.Conclusions: LNT-Se had a good anti-acute mononuclear leukemia effect in vitro and in vivo, and could be absorbed by cells, and the primary target organelle was lysosome. Both in vivo and in vitro experiments suggested that LNT-Se was less toxic. LNT-Se had antioxidant activity and might exert anti-AML-M5 activity through TNF, MAPK and NF-κB pathways. These results indicated that LNT-Se could be used as an effective anti-AML-M5 drug. DisclosuresNo relevant conflicts of interest to declare.

The Impact of Hypomethylating Agents and BCL-2 Inhibitor on HLA Expression on THP-1 Cells

Note: BP, MV and LG, KG contributed equallyBackgroundRelapsed acute myeloid leukemia (AML) remains the most common reason for allogeneic hematopoietic cell transplant (HCT) failure. Thus, understanding AML immune escape mechanism is important for improving the odds of curing HCT patients with AML. Downregulation of HLA Class I and II expression by AML is one of the potential immune escape mechanisms. Therefore, treatment to restore HLA surface expression is crucial to prevent and treat relapse. Endogenous cytokines, such as IFN-γ, have been shown to stimulate HLA expression but are poorly tolerated by patients. However, two hypomethylating agents (HMA), decitabine (Dec) and azacitadine (Aza), that are routinely used in AML treatment are known to augment HLA expression. For AML, HMAs are often combined with venetoclax (Ven), a drug that blocks the anti-apoptotic B-cell lymphoma-2 (Bcl-2) protein. Thus, while HMAs have been reported to increase HLA expression, what is unknown is whether these agents impact individual HLA loci differently and whether Ven has any impact on HLA expression. To address these questions, we treated the THP-1 cell line with Dec, Aza or Ven and measured changes in cell-surface expression of HLA proteins by flow cytometry using locus-specific HLA mAbs.MethodsTHP-1 cells were incubated with IFN-γ (500 U/mL), Aza (2µM), Dec (5µM), or Ven (30nM) for 48 hours (drug concentrations were determined by earlier titration experiments). THP-1 cells are a monocytic cell line, derived from the peripheral blood of a childhood case of acute monocytic leukemia (M5 subtype), that express HLA Class I and HLA-DR but not HLA-DQ or -DP under basal conditions, although they are inducible by IFN-γ. Thus, the induction of HLA Class II expression by IFN-γ serves as a positive control. Isotype controls were included to measure background. Data is presented as the difference in MFI (delta MFI) between cells treated with a drug and those treated with diluent only.ResultsTreatment of THP-1 cells with either IFN-γ or Dec led to increases in Class I HLA-A, -B & -C (Figure 1) compared to untreated cells (a mean fold increase of 1.4 and 1.2, respectively). Notably, Aza did not stimulate additional HLA-C expression and induced less of an increase in HLA-A & -B expression (an increase of 1.1-fold) than IFN-γ or Dec. Treatment of THP-1 cells by Ven did not induce a change in HLA Class I expression.For Class II, IFN-γ or Dec increased HLA-DR, -DQ and -DP expression in comparison to untreated cells (Figure 1). IFN-γ induced greater HLA-DR expression compared to Dec (an increase of 2.3-fold and 1.5-fold, respectively), and both stimulated similar increases in HLA-DQ (increases of 1.5-fold and 1.4-fold, respectively) & -DP (increases of 1.9-fold and 1.5-fold, respectively). However, treatment of cells with either Aza or Ven did not lead to changes in HLA Class II expression.DiscussionPrevious studies have illustrated the ability of IFN-γ to induce HLA Class II expression in THP-1 cells, however, data for Dec to induce HLA Class II expression was unconfirmed. We report differences in the degree to which IFN-γ and Dec are capable of stimulating HLA-DR with IFN-γ being more potent. The inability of Aza to induce HLA Class II expression in THP-1 cells may be related to the differing drug activating pathways of the two HMAs. Indeed, there are conflicting reports as to whether Aza can stimulate HLA Class II expression. Though Ven treatment of THP-1 cells did not impact HLA expression, because it is given with HMAs, it remains to be seen what effect these drugs may have on HLA expression when administered together. Additional studies to confirm these observations in patient-derived AML blasts are ongoing.ConclusionWe report that HMAs increased expression of HLA-A, -B, & -C loci and Dec but not Aza stimulated HLA-DR, -DQ, and -DP expression in THP-1 cells. Given these data, Dec may be superior in increasing HLA Class II expression post-HCT. [Display omitted] DisclosuresMarcucci: Abbvie: Other: Speaker and advisory scientific board meetings; Agios: Other: Speaker and advisory scientific board meetings; Novartis: Other: Speaker and advisory scientific board meetings. Al Malki: Neximmune: Consultancy; CareDx: Consultancy; Jazz Pharmaceuticals, Inc.: Consultancy; Rigel Pharma: Consultancy; Hansa Biopharma: Consultancy.

Identification of Potent and Selective Inhibitors of Fat Mass Obesity-Associated Protein Using a Fragment-Merging Approach.

Fat mass obesity-associated protein (FTO) is a DNA/RNA demethylase involved in the epigenetic regulation of various genes and is considered a therapeutic target for obesity, cancer, and neurological disorders. Here, we aimed to design novel FTO-selective inhibitors by merging fragments of previously reported FTO inhibitors. Among the synthesized analogues, compound 11b, which merges key fragments of Hz (3) and MA (4), inhibited FTO selectively over alkylation repair homologue 5 (ALKBH5), another DNA/RNA demethylase. Treatment of acute monocytic leukemia NOMO-1 cells with a prodrug of 11b decreased the viability of acute monocytic leukemia cells, increased the level of the FTO substrate N6-methyladenosine in mRNA, and induced upregulation of MYC and downregulation of RARA, which are FTO target genes. Thus, Hz (3)/MA (4) hybrid analogues represent an entry into a new class of FTO-selective inhibitors.

Synthesis of 1-Aryloxymethyl-2,4,5-trifluorobenzene Analogs and Their Inhibitory Activity of CCL2 Expression

CCL2 overexpression is associated with macrophage recruitment during immune response and poor patient prognosis in various cancers, such as prostate, breast, pancreatic, and liver cancer. For developing CCL2 inhibitors, 1-aryloxymethyl-2,4,5-trifluorobenzene analogs were designed and synthesized by concise synthetic route including SN2 reaction, reduction, and oxime formation reaction. Furthermore, it was confirmed that they inhibited CCL2 production induced by 27-hydroxycholesterol through reverse transcription PCR in the human acute monocytic leukemia cell line (THP-1). Among them, N-((2-(2,4,5-trifluorobenzyloxy)naphthalene-1-yl)methyl)hydroxylamine (4) exhibited the highest CCL2 transcription inhibition activity.

COEXISTENCE OF ACUTE MONOBLASTIC LEUKEMIA IN A CHRONIC LYMPHOCYTIC LEUCEMIA: CASE REPORT

Chronic lymphocytic leukemia (CLL) is associated with an increased risk of other malignancies, such as skin and. second hematological malignancies. In the majority of cases, this usually involves a disease transformation to an aggressive non-Hodgkin lymphoma, multiple myeloma or prolymphocytic lymphoma. CLL patients can rarely develop acute myeloid leukemia (AML). We have reported a case in which there was simultaneous presentation of AML and CLL. A 78-year-old man presented with tiredness and mucosal bleeding. Laboratory evaluation revealed a white blood count of 220,970 μ/l with 4% of blasts,69% lymphocytes and 20% monocytes, hemoglobin of 6.7 g/dl, platelet count of 29,000 μ/l. A bone marrow aspirate revealed markedly hypercellular bone marrow with 21% blasts and promonocytes and 47% mature lymphocytes. A peripheral blood flow cytometry demonstrated a mature CD5 and CD23 positive B-cell population expressing CD19, low-density CD 20, CD11c, CD25, CD81, CD39 and lambda light chains at 53.02% of lymphocytes, consistent with B-CLL.; In addition, a second population showed CD33, CD13 low-density, CD11b, CD64, CD14, CD36, HLA-DR, CD56, CD123, CD11c, CD38 and CD9 positives in 39.35% of immature monocytes, consistent with acute monoblastic leukemia. Cytogenetics on bone marrow aspirate showed 48,XY, +4,+8[18] / 46,XY [2]. The present report highlights the rare possibility of the development of a myeloid malignancy in a patient with lymphoid malignancy. Most of these cases more frequently present as a secondary event in patients receiving chemotherapeutic agents for CLL.Knowledge of this rare association is the key to timely and accurate diagnosis, particularly in patients with atypical presentation. Existing literature demonstrates that AML may also develop concurrently or even after the diagnosis of treatment-naïve CLL patients. Further studies of such patients have potential to provide insight into the pathogenesis of myeloid as well as lymphoid malignancies.

Bilateral parotid gland enlargement as a presenting manifestation of relapsed pediatric acute myeloid leukemia

Parotid gland involvement is a rare presentation of Acute Myeloid Leukemia (AML). We report a 10-year-old girl of acute myelomonocytic leukemia with normal cytogenetics and positivity for inversion 16, who after completion of first consolidation of the BFM 2004 AML protocol, presented with bilateral parotid gland enlargement. Bone marrow examination was suggestive of a relapse. Fine needle aspiration of the parotid gland showed presence of myeloblasts. Patient was given FLAG-IDA chemotherapy, with which the parotid swellings rapidly regressed and she achieved a remission and was planned for allogenic bone marrow transplantation. Acute myelomonocytic and monoblastic leukemias are known to be associated with tissue infiltration. However, exocrine gland involvement such as parotid enlargement is rare and is associated with a poor prognosis as was seen in our patient who despite having inversion 16 positivity had poor disease outcome. Keywords: acute myeloid leukemia; parotid gland enlargement; granulocytic sarcoma.

Chronic monocytic leukemia with transformation into acute monocytic leukemia. Clinical case

Background. Chronic myelomonocytic leukemia (CML) is rarely diagnosed and it is 1 per 100 thousand adults annually, in the United States - in 4 per million people, which is about 1100 cases per year. This disease is more common for men over 60. Results. A clinical case of a rare long-term course of myelodysplastic chronic myelomonocytic leukemia (MDCMML) in a middle-aged woman with rapid transformation into acute monocytic leukemia (AMoL-M5v) with atypical fulminant course is presented. Changes in the blood test were identified accidentally during a routine examination. A retrospective analysis of the course of the patient's disease, anamnesis made it possible to draw attention to the severe course of vasculitis of unknown etiology, with a predominant lesion of the skin of the lower limbs, which required inpatient treatment (19 years ago); skin lesions in the form of transient erythema, spotty eruptions for more than 10 years, moderate cervical lymphadenopathy. According to the WHO criteria, the morphological data of the bone marrow puncture corresponded to the MD of the CML. The long course of the disease without an obvious clinical picture, neutrophil dysplasia, myeloid proliferation was atypical, which did not exclude the presence of previous oligomonocytic CML in the patient. A detailed picture of the disease appeared after a viral infection, bronchitis, antibiotic therapy. In the absence of an increase in the number of blasts in the bone marrow, in a few of them normal Auer's sticks were detected, which, according to the literature, is a rarity in CML and an unfavorable prognostic factor of rapid transformation into acute myeloid leukemia. Conclusion. Not typical for the course of acute monocytic leukemia in this case were the absence of significant blastemia and severe suppression of normal hematopoiesis with pronounced extramedular manifestations. There was febrile fever, hyperplasia of the gums, tonsils with ulcerative-necrotic changes in the oral mucosa, an increase in cervical lymph nodes in the form of packets up to 2 cm in diameter with signs of sarcomatous growth. Attention was drawn to the progression of skin lesions, which was prognostically unfavorable. Notable was the development of severe hemorrhagic syndrome without severe thrombocytopenia, significant changes in the coagulogram, as a manifestation of early severe coagulopathy. There was a spread of erythematous elements on the skin with itching, not controlled by antihistamines and corticosteroid drugs (maculopapular rashes of a pink-cyanotic color, in places of a confluent nature, small-point hemorrhages like vasculitis over the entire surface of the skin).

MiR‑4792 regulates inflammatory responses inCryptococcus neoformans‑infected microglia.

Investigating the factors that influence the inflammatory response of microglial cells is crucial for understanding the pathogenesis of cryptococcal meningitis (CM). MicroRNAs (miRNAs/miRs) play an important role in inducing host defenses and activating the immune response during microbial infection; however, the regulatory mechanisms of miRNAs in cryptococcal meningitis remain poorly defined. In a previous study, the authors assessed the miRNA profiles of THP‑1 (human acute monocytic leukemia cells) cells following Cryptococcus neoformans (C.neoformans) infection. In the present study, it was found that miR‑4792 expression was downregulated in BV2 cells infected with C.neoformans, whilst that of its target gene, epidermal growth factor receptor (EGFR), was upregulated. Infected cells in which miR‑4792 was overexpressed exhibited a decreasedEGFR transcript expression, reduced mitogen‑activated protein kinase (MAPK) signaling and a decreased secretion of inflammatory cytokines. In addition, following antifungal treatment in patients with cryptococcal meningitis, the levels of miR‑4792 in the cerebrospinal fluid significantly increased, whilst the expression of EGFR significantly decreased. In addition, receiver operator characteristic analysis revealed miR‑4792 (AUCROC=0.75) and EGFR (AUCROC=0.79) as potential diagnostic markers in patients with cryptococcal meningitis.

FNF-12, a novel benzylidene-chromanone derivative, attenuates inflammatory response in in vitro and in vivo asthma models mediated by M2-related Th2 cytokines via MAPK and NF-kB signaling.

This study evaluates a novel benzylidene-chromanone derivative, FNF-12, for efficacy in in vitro and in vivo asthma models. Rat basophilic leukemia (RBL-2H3) and acute monocytic leukemia (THP-1)-derived M2 macrophages were used. Human whole blood-derived neutrophils and basophils were employed. Flow cytometry was used for studying key signalling proteins. Platelet activation factor (PAF)-induced asthma model in guinea pigs was used for in vivo studies. The chemical structure of FNF-12 was confirmed with proton-nuclear mass resonance (NMR) and mass spectroscopy. FNF-12 controlled degranulation in RBL-2H3 cells with an IC50 value of 123.7nM and inhibited TNF-α release from these cells in a dose-responsive way. The compound effectively controlled the migration and elastase release in activated neutrophils. IC50 value in the FcεRI-basophil activation assay was found to be 205nM. FNF-12 controlled the release of lipopolysaccharide (LPS)-induced interleukin-10, I-309/CCL1 and MDC/CCL22 in THP-1 derived M2 macrophages. The compound suppressed LPS-induced mitogen activated protein kinase (MAPK)-p-p38 and nuclear factor kappa B(NF-kB)-p-p65 expression in these cells. A dose-dependent decrease in the accumulation of total leucocytes, eosinophils, neutrophils and macrophages was observed in PAF-induced animal models. FNF-12 was able to control the inflammatory responses in in vitro and in vivo asthma models, which may be driven by controlling M2-related Th2 cytokines via MAPK and NF-kB signaling.

Antiviral, Cytotoxic, and Antioxidant Activities of Three Edible Agaricomycetes Mushrooms: Pleurotus columbinus, Pleurotus sajor-caju, and Agaricus bisporus.

In this study, we investigated aqueous extracts of three edible mushrooms: Agaricus bisporus (white button mushroom), Pleurotus columbinus (oyster mushroom), and Pleurotus sajor-caju (grey oyster mushroom). The extracts were biochemically characterized for total carbohydrate, phenolic, flavonoid, vitamin, and protein contents besides amino acid analysis. Triple TOF proteome analysis showed 30.1% similarity between proteomes of the two Pleurotus spp. All three extracts showed promising antiviral activities. While Pleurotus columbinus extract showed potent activity against adenovirus (Ad7, selectivity index (SI) = 4.2), Agaricus bisporus showed strong activity against herpes simplex II (HSV-2; SI = 3.7). The extracts showed low cytotoxicity against normal human peripheral blood mononuclear cells (PBMCs) and moderate cytotoxicity against prostate (PC3, DU-145); colorectal (Colo-205); cecum carcinoma (LS-513); liver carcinoma (HepG2); cervical cancer (HeLa); breast adenocarcinoma (MDA-MB-231 and MCF-7) as well as leukemia (CCRF-CEM); acute monocytic leukemia (THP1); acute promyelocytic leukemia (NB4); and lymphoma (U937) cell lines. Antioxidant activity was evaluated using 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical scavenging, 2,2′-Azinobis-(3-Ethylbenzthiazolin-6-Sulfonic Acid) ABTS radical cation scavenging, and oxygen radical absorbance capacity (ORAC) assays. The three extracts showed potential antioxidant activities with the maximum activity recorded for Pleurotus columbinus (IC50 µg/mL) = 35.13 ± 3.27 for DPPH, 13.97 ± 4.91 for ABTS, and 29.42 ± 3.21 for ORAC assays.

Effect and Involved Mechanism of RSL3-induced Ferroptosis in Acute Leukemia Cells MOLM13 and Drug-resistant Cell Lines

To investigate the effect and involved mechanism of RSL3 on ferroptosis action in acute leukemia cells MOLM13 and its drug-resistant cells. After MOLM13 treated with RSL3, CCK-8 assay was performed to detect cell viability, flow cytometry was used to detect the reactive oxygen species (ROS) level of the cells, RT-qPCR and Western blot were used to detect the expression of glutathione peroxidase 4 (GPX4). After MOLM13/IDA and MOLM13/Ara-C, the drug-resistant cell lines were constructed, the ferroptosis induced by RSL3 was observed. Bone marrow samples were collected from patients with acute monocytic leukemia. RT-qPCR and Western blot were performed to detect the expression of related genes and proteins involved in ferroptosis pathway. RSL3 significantly inhibited the cell viability of MOLM13 and increased the intracellular ROS level, which were partially reversed by ferrostatin-1. The mRNA and protein expression of GPX4 decreased in MOLM13 treated with RSL3. RSL3 inhibited the viability of MOLM13/IDA and MOLM13/Ara-C cells more strongly than that of non-drug resistant cells, also increased the intracellular ROS level . The cytotoxic effects were partially reversed by ferrostatin-1. The mRNA and protein expressions of GPX4 in MOLM13/IDA and MOLM13/Ara-C cells were higher than those in non-drug resistant cells. The mRNA and protein levels of GPX4 in bone marrow of relapsed/refractory acute mononuclear leukemia patients were higher than those of ordinary acute mononuclear leukemia patients. RSL3 can induce non-drug resistant cells MOLM13 ferroptosis by inhibiting GPX4 activity. MOLM13/IDA and MOLM13/Ara-C are more sensitive to RSL3 compared with non-drug resistant cells MOLM13, which may be caused by the differences in GPX4 expression. The expressions of GPX4 mRNA and protein in relapsed/refractory acute mononuclear leukemia are higher than those in ordinary acute mononuclear leukemia.

Hemoglobin scavenger receptor (cluster of differentiation 163) role in acute leukemia

Background Cluster of differentiation 163 (CD163) is a biomarker correlated with several normal and pathological states. Aim This work was carried out to evaluate the expression of CD163 in patients with acute leukemia. Patients and methods The study was carried out on 50 participants divided into three groups: 10 apparently normal healthy individuals, 30 patients with acute myeloid leukemia (AML), and 10 patients with acute lymphoid leukemia. All participants were subjected to a thorough history and clinical examination. Becton–Dickinson Calibur FACScan color multiparameter flow cytometry was used for the detection of CD163 expression in patients with acute leukemia. Results There was a significant difference in CD163 expression between AML and acute lymphoid leukemia (F=7.83) (P=0.001). CD163 expression was not observed in patients with acute lymphoblastic leukemia. However, it was expressed in 14 patients with acute myeloblastic leukemia. Five patients were diagnosed as M4, and all of them (100%) showed positive expression of CD163. Eight patients were diagnosed as M5, and all of them (100%) showed positive expression of CD163. However, CD163 was expressed in only one case among 17 patients with AML subtypes other than M4/M5. There was a significant difference between monocytic and nonmonocytic leukemic patients regarding CD163 expression (P<0.001). There was a strong correlation between CD163 and other markers predominantly found in monocytic leukemia such as CD14 (r=0.8) (P<0.001), CD15 (r=6.43) (P<0.001), and CD64 (r=0.82) (P<0.001). Conclusion CD163 was exclusively expressed on the monocytic and myelomonocytic leukemia, so it can be used for the diagnosis of the monocytic type of AML. Although it cannot be used as a prognostic marker, it could be a novel immunotherapeutic intervention for acute monocytic and myelomonocytic leukemia.

CDX2 expression in the hematopoietic lineage promotes leukemogenesis via TGFβ inhibition.

The intestine‐specific caudal‐related homeobox gene‐2 (CDX2) homeobox gene, while being a tumor suppressor in the gut, is ectopically expressed in a large proportion of acute leukemia and is associated with poor prognosis. Here, we report that turning on human CDX2 expression in the hematopoietic lineage of mice induces acute monoblastic leukemia, characterized by the decrease in erythroid and lymphoid cells at the benefit of immature monocytic and granulocytic cells. One of the highly stimulated genes in leukemic bone marrow cells was BMP and activin membrane‐bound inhibitor (Bambi), an inhibitor of transforming growth factor‐β (TGF‐β) signaling. The CDX2 protein was shown to bind to and activate the transcription of the human BAMBI promoter. Moreover, in a leukemic cell line established from CDX2‐expressing mice, reducing the levels of CDX2 or Bambi stimulated the TGF‐β‐dependent expression of Cd11b, a marker of monocyte maturation. Taken together, this work demonstrates the strong oncogenic potential of the homeobox gene CDX2 in the hematopoietic lineage, in contrast with its physiological tumor suppressor activity exerted in the gut. It also reveals, through BAMBI and TGF‐β signaling, the involvement of CDX2 in the perturbation of the interactions between leukemia cells and their microenvironment.

RNA Editing By APOBEC3A Confers Dynamic Mutational Load in Monocytic Leukemias

Background: Although genomic mutations play an important role in the initiation or progression of myeloid leukemias, the role of mutations at the transcript level is less well understood. Myeloid neoplasms have recurrent somatic mutations in genes for pre-mRNA splicing, cohesin complex, histone modification, and DNA methylation. However, the number of somatic mutations identified in myeloid neoplasms (average 9-13) is usually lower than that of solid tumors. Recent analyses of baseline transcriptome sequences in various cancers have revealed abundant adenosine to inosine (A>I) RNA editing events, mainly at intronic and untranslated regions of genes. The role of condition-specific RNA editing in cancer, especially of the cytosine to uracil (C>U) type, however, remains essentially unknown. We recently discovered antiviral restriction enzyme APOBEC3A (A3A) cytidine deaminase as a novel cellular site-specific C>U RNA editing enzyme in monocytes. RNA editing by A3A is dynamically induced by interferons and/or severe hypoxia in monocytes, resulting in stop codon gains or missense changes in transcripts of scores of genes. The effect of hypoxia in inducing RNA editing by A3A can be mimicked by inhibition of mitochondrial respiration. Whether A3A-mediated RNA editing occurs in monocytic leukemias, including acute monocytic leukemia (AMoL) and chronic myelomonocytic leukemia (CMML), is unknown. We hypothesize that dynamic C>U RNA editing by A3Acontributes to mutational load of leukemic cells under stressful conditions of hypoxia and interferon exposure. In this study, we examined transcript sequences of monocytic leukemias for evidence of inducible RNA editing. We also examined the role of mitochondrial respiratory inhibition, which induces RNA editing by A3A, in growth of leukemic progenitors of CMML.Methods: Monocytic leukemia samples are obtained from the RPCI Leukemia Bank. Standard diagnostic information included classification (WHO 2008 and FAB), fraction of monocytic component (mature monocytes, promonocytes or monoblasts by flow cytometry and/or aspirate differential count), cytogenetic analyses (conventional karyotype and targeted FISH), and survival data. Leukemic BM mononuclear cells (BM-MNC) prepared by the Ficoll gradient method are thawed and cultured in hypoxia (1% O2) with interferon type 1 (IFN-1). DNA and RNA are isolated. AMoL samples are subjected to nextgen high throughput exome and RNA sequencing. Once variants from DNA and RNA of corresponding samples are obtained, the set difference of variants is extracted to keep those detected on RNA samples. We also examined 3 CMML bone marrow samples for evidence of hypoxia-inducible RNA editing by analyzing selected RNA editing sites. Colony forming unit (CFU) assays are also performed in CMML samples.Results: We find that the A3A gene is robustly expressed inboth in AMoL and CMML. Hypoxia (with or without IFN-1) induced A3A-mediated RNA editing in AMoL and CMML samples. We previously showed that several RNA edited genes by A3A in normal monocytes function in recurrently altered pathways in myeloid leukemias. Sanger sequencing confirmed RNA editing of such genes in monocytic leukemia samples as well (Table 1), suggesting that epitranscriptomic sequence changes may contribute to molecular defects driving leukomogenesis. RNA seq analysis of AMoL samples (n=3) showed that dozens of genes acquire nonsense or missense mutations at the transcript level under the conditions of severe hypoxia and IFN-1. CFU assays of CMML samples showed that inhibition of mitochondrial respiration, which triggers A3A-mediated RNA editing, inhibits the growth of progenitor cells (Figure 1).Conclusions: We show that A3A-mediated RNA editing is active in monocytic leukemias and targets genes functioning in pathways recurrently altered in myeloid leukemias. RNA editing by A3A may contribute to growth arrest and survival of leukemic progenitors under certain stress conditions. To our knowledge, these results provide the first experimental evidence that conditional RNA editing by A3A confers substantial mutational load in myeloid leukemias. Such RNA editing events will dynamically increase the total mutational load in monocytic leukemias, and may create neo-antigens for immune therapy by checkpoint inhibition. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Immunotoxin Targeting Cell-Bound MUC1 on Myelomonocytic and Monocytic Leukemic Cells

Cell surface molecules aberrantly or overexpressed by myeloid leukemic cells represent potential disease-specific therapeutic targets for antibodies. MUC1, a polymorphic type I high molecular weight glycoprotein represents such a target. MUC1 is cell surface molecule with extracellular, transmembrane, and cytoplasmic domains; the cytoplasmic tail participates in intracellular signaling. Cleavage of MUC1 yields two unequal chains: a large extracellular alpha subunit bound in strong non-covalent interaction to a smaller beta subunit containing the transmembrane and cytoplasmic domains. Essentially all anti-MUC1 antibodies reported to date target the highly immunogenic MUC1 alpha chain. Because the MUC1 alpha chain binds the cell only intermittently in an ‘on-and-off’ manner, agents directed against the alpha chain will not effectively target MUC1+ cells. In contrast, the MUC1 SEA domain represents a stable structure fixed to the cell surface at all times. We therefore generated mAbs that specifically recognize the cell-bound MUC1 SEA domain. One of them, a partially humanized murine mAb termed DMB-5F3 was used to examine the expression of MUC1 on leukemic cells by flow cytometry. DMB-5F3 was found to strongly bind not all AML, but rather a specific set of AML subtypes: Chronic Myelomonocytic Leukemia (CMML); Acute Myelomonocytic and Monocytic Leukemia (AML-M4 and AML-M5), and Juvenile Myelomonocytic Leukemia (JMML). Other AML subtypes did not express MUC1. To test the cytotoxic ability of anti-MUC1 antibodies against MUC-expressing AML, we generated a humanized, dimeric single-chain DMB-5F3 scFv fragments fused to recombinant gelonin, a type I ribosome-inactivating toxin which itself is incapable of cell binding or internalization into mammalian cells to which a 6xHis-tag was added. The anti-MUC1 scFv-toxin cytometrically identified using an anti-6xHis-tag was found to bind MUC1 positive cell line. We examined the cytotoxic effects of anti MUC1 scFV-toxin using the MUC-1 positive AML cell lines MV411 and MOLM14 (monocytic leukemic subtype). MUC1 scFV-toxin was found to be highly cytotoxic on MUC1+ AML cell lines, resulting in direct cell death. Our data strongly support the use of an anti-MUC 1 antibody-drug conjugate to selectively target and inhibit the growth of MUC1-expressing AML. DisclosuresNo relevant conflicts of interest to declare.